| Aims The aims of the study were to investigate the changes of gastric motility and interstitial cells of cajal in the gastric antrum of diabetic rats induced by STZ.Methods20male Sprague Dawley (SD) rats were randomly divided into two groups, the diabetes group (DM proup, n=10) was established diabetes model though60mg/kg streptozotocin intraperitoneal injection; the remaining10rats were as normal control group (normal control group, n=10). Blood glucose and body weight of the two groups was recorded before modeling, after model,2weeks,4weeks and6weeks after the model, respectively. After6weeks of the model, the change of gastric motility in two groups of rats was evaluated though phenol red irrigation method to test gastric emptying and organ bath technique to test the circular muscle contraction of gastric antrum; immunohistochemistry, RT-PCR and Western blot were used to evaluate the expression of c-kit in gastric antrum; transmission electron microscope (TEM) was applied to detect the ultrastructural damage of ICC.Results (1) There was no significant difference in blood glucose in rats of the two groups before the model; after model and2weeks,4weeks and6weeks after the model, blood glucose of diabetic rats was increased significantly compared with the normal group;(2) There was no significant difference in body weight in rats of the two groups before the model; after model and2weeks,4weeks and6weeks after the model, the body weight of rats in diabetic group was decreased significantly than that in the normal group;(3) Gastric emptying of diabetic rats was delayed significantly compared to the normal group (0.57±0.06%vs0.73±0.02%, P=0.018);(4) before and after selective destruction of ICC, contraction of muscle strips in diabetic group were decreased significantly compared with normal group;(5) RT-PCR showed that the amount of c-kit mRNA expression in gastric antrum of diabetic rats was significantly decreased compared to the normal group (0.024±0.001vs1±0.095, P=0.000);(6) Western blot study showed that the expression of c-kit protein in gastric antrum of diabetic rat was significantly lower than the normal group;(7) In normal group, c-kit positive cells in gastric antrum were mainly distributed in the myenteric muscle layer, intramuscular layer and the submucosa layer, the expression of c-kit positive cells in each layer was reduced in diabetic group;(8) The ultrastructure of ICC in diabetic group than the normal group showed cells with reduced organelle, mitochondria and endoplasmic reticulum swelling.Conclusion Diabetic rats showed gastric emptying obviously delayed, antral contraction reduced, accompanying with the loss and damage of ICC. Objective To investigate the effects of SCF/c-kit signal pathway and SMC on the change of ICC in diabetic gastroparesis rats.Methods The expression of SCF, a-SMA, mysionl1protein were detected by Western blot; RT-PCR was applied to detect the mRNA expression of SCF, a-SMA; the detection of smooth muscle cells and ICC were used double immunofluorescence of a-SMA and c-kit. Transmission electron microscopy was applied to observe the ultrastructure change of SMC.Results (1) Western blot showed that compared with the normal control group, the expression of SCF protein in gastric antrum of diabetic rats was significantly decreased, a-SMA expression was not significantly changed, while mysion11expression was significantly lower than the normal control group;(2) RT-PCR showed that compared to the normal control group, the expression of SCF mRNA in gastric antrum of diabetic rats was significantly lower, but there was no obvious change in the expression of a-SMA;(3) double immunofluorescence results showed that in the normal control group, the c-kit+cells were located among a-SMA+cells, more ICCs were distributed in the muscle layer and the inner layer, and c-kit+cells in diabetic group were significantly reduced;(4) the control group, SMC was spindle shaped, the nucleus was elongated oval, nuclear heterochromatin distribution was uniform, cytoplasmic dense patches and dense body were obviously observed, filament was clearly visible, the mitochondria were rich. The diabetic group, SMC was arranged in disorder with irregular nucleus, the chromatin was massive, dark staining, many large cytoplasm lysis vacuoles were distributed intracellular, dense bodies and myofilament were decreased, mitochondrial was swelling, vacuolar degeneration, dissolution.Conclusion In diabetes rats, SCF/c-kit signal pathway was down regulated, there were not significantly reduced of SMC, but the structure had been significant damaged. Objective To observe that long pulse gastric electrical stimulation could effectively improve the gastric motility of diabetic gastroparesis rats and the effect on ICC in gastric antrum, and further investigate the underlying mechanism.Methods40male Sprague Dawley (SD) rats were successfully prepared for gastric electrical stimulation model. Two weeks after postoperative recovery, diabetic rat model was prepared using streptozotocin (STZ) by intraperitoneal injection of60mg/kg system solution. Rats were randomly divided into diabetic+sham stimulation group (DM+SGES), diabetic+true stimulation group (DM+GES). Sham stimulation group (DM+SGES) was only buried electrode, not given electrical stimulation, while true stimulation group (DM+GES) was given different parameters of long pulse stimulation after electrode buried. The following parameters: GES15.5cpm,100ms,4mA; GES25.5cpm,300ms,4mA; GES35.5cpm,550ms,2mA. Stimulation was chronic,30min/days, a total of6weeks. Blood glucose and body weight were recorded in rats before modeling, after model, after2weeks,4weeks and6weeks, respectively. After6weeks of model, gastric emptying by gastric perfusion using phenol and circular muscle contraction by organ bath technique was used to evaluate gastric motility of rats. The gastric antrum tissue of rats in each group taken, the expression of c-kit, SCF, a-SMA, myosinll in gastric antrum tissue were evaluated with the application of Western blot and RT-PCR technique; immunohistochemistry was used to observe the expression of ICC; double immunofluorescence was used to observe the expression of ICC and SMC; ultra microstructure of ICC and SMC was detected by transmission electron microscopy.Results (1) There was no significant difference of blood glucose in each group before the model; after modeling and after model2weeks,4weeks and6weeks, the true and false stimulation group and diabetes group had no obvious difference;(2) There was no significant difference of body weight in each group before the model; after modeling and after model2weeks and4weeks, body weight of true stimulated rats was not changed compared with diabetic group; after model6weeks, body weight of true stimulated rats compared with diabetic group was increased significantly;(3) Gastric emptying in DM group was significantly reduced than in the normal control group, after given the different parameters of GES, gastric emptying were obviously improved, especially GES2and GES3;(4) Antral contraction of circular muscle strips in DM group was significantly decreased than in the normal control group, after given the different parameters of GES, the contraction of antral muscle strips were obviously improved;(5) Western blot study showed that after given long pulse gastric electrical stimulation, the expression of c-kit, SCF and myosin11protein in diabetic group was significantly increased compared with DM group, whereas the expression of a-SMA was not significantly changed;(6) RT-PCR showed that the expression of c-kit, SCF mRNA in gastric antrum of true stimulated rats was significantly increased compared with DM group, while a-SMA expression was not significantly changed;(7) immunohistochemical technique showed that c-kit positive cells in the true stimulation group were distributed in the muscle layer, muscle layer and submucosa of gastric antrum;(8) Double immunofluorescence showed after given gastric electrical stimulation, more c-kit+cells among a-SMA+cells in diabetic rats were observed, namely more ICC distributed in the myenteric layer and the muscle layer;(9) The ultrastructure of ICC in the true stimulation group compared with diabetic group showed intact cell membrane, obviously decreased nuclear heterochromatin, mitochondrial and endoplasmic reticulum not swelled significantly; SMC in the true stimulation group was arranged in neat rows, fusiform, nuclear heterochromatin distributed evenly, without larger cytoplasmic vacuoles, dense bodies and myofilament rich, organelles without obvious swelling and expansion.Conclusion Long pulse gastric electrical stimulation could increase the body weight of diabetic rats, and effectively improve the delayed gastric emptying and imperfect antral contractility; long pulse gastric electrical stimulation also significantly increased ICC expression in gastric antrum of diabetic rats and improve the pathological injury of ICC, upregulate the expression of SCF/c-kit signal pathway, and improve the structure damage of SMC. |
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